Soil bacteria of the genus Rhizobium, a member of the family Rhizobiaceae, are capable of infecting plants and inducing a highly differentiated structure, the root nodule, within which atmospheric nitrogen is reduced to ammonia by the bacteria. The host plant is most often of the family Leguminosa. Previously, Rhizobium species were informally classified in two groups, either as "fast-growing" or "slow-growing" to reflect the relative growth rates in culture. The group of "slow-growing" rhizobia has recently been reclassified as a new genus, Bradyrhizobium (Jordan, D.C. (1982) International Journal of Systematic Bacteriology 32:136). The fast-growing rhizobia include Rhizobium trifolii, R. meliloti, R. leguminosarum and R. phaseolus. These strains generally display a narrow host range. Fast-growing R. japonicum which nodulate wild soybeans, Glycine max cv. Peking and siratro, and fast-growing members of the cowpea Rhizobium display broader host range. R. japonicum strains form only ineffective nodules on commercial soybean cultivars. The fast-growing R. japonicum strains are now designated R. fredii (Scholla and Elkan (1984) Int'l. Journal Systematic Bacteriol. 34:484-486). The slow-growing Bradyrhizobium include the commercially important soybean nodulating strains Bradyrhizobium japonicum (i.e., USDA 110 and USDA 23), the symbiotically promiscuous rhizobia of the "cowpea group", including Bradyrhizobium sp. (Vigna) and Bradyrhizobium sp. Parasponia (formerly Parasponia Rhizobium) which nodulates the non-legume Parasponia, as well as a number of tropical legumes including cowpea and siratro. Within the species B. japonicum, a number of distinct serogroups represented by USDA110 and USDA123, for example, are recognized. Strains belonging to a serogroup have been found to display quantitatively different nodulation or symbiotic properties when compared to strains of other serogroups. For example, B. japonicum USDA110 are more effective for nitrogen fixation than USDA123 strains, but USDA123 strains appear to be more competitive for infection and nodule occupancy when compared to USDA110 strains.
Nodulation and development of effective symbiosis is a complex process requiring both bacterial and plant genes. Several recent reviews of the genetics of the Rhizobium-legume interaction are found in Broughton, W.J., ed. (1982) Nitrogen Fixation. Volumes 2 and 3 (Clarendon Press, Oxford); Puhler, A. ed. (1983) Molecular Genetics of the Bacteria-Plant Interaction (Springer-Verlag, Berlin); Szalay, A.A. and Leglocki, R.P., eds. (1985) Advances in Molecular Genetics of the Bacteria-Plant Interaction (Cornell University Publishers, Ithaca, New York); Long, S.R. (1984) in Plant Microbe Interactions Volume 1, Kosuge, T. and Nester, E.W. eds. (McMillan, New York) pp. 265-306; and Verma, D.P.S. and Long, S.L. (1983) International Review of Cytology (Suppl. 14), Jeon, K.W. (ed.), Academic Press, p. 211-245.
In the fast-growing species, the genes required for nodulation and nitrogen fixation are located on large Sym (symbiotic) plasmids. Although the process of recognition, infection and nodule development is complex, it appears that at least for the fast-growing rhizobia relatively few bacterial genes are directly involved and these are closely linked on the Sym plasmid. For example, a 14 kb fragment of the Rhizobium trifolii Sym plasmid is sufficient to confer clover-specific nodulation upon a Rhizobium strain cured of its Sym plasmid, as well as on an Aorobacterium strain which does not normally nodulate plants (Schofield et al., (1984) Plant Mol. Biol. 3:3-11). Nodulation and nitrogenase genes are also localized on symbiotic plasmids in R. leguminosarum (Downie et al. (1983) Mol. Gen. Genet. 190:359-365) and in R. meliloti (Kondorosi et al. (1984) Mol. Gen. Genet. 193:445-452).
Fine structure genetic mapping has been used to locate individual nodulation genes in fast-growing rhizobia. Transposon mutagenesis, most often using the transposon Tn5, has identified about 10 nodulation genes associated with non-nodulation, delayed nodulation and altered host range phenotypes (Djordjevic et al. (1985) Mol. Gen. Genet. 200:263-271; Downie et al. (1985) Mol. Gen. Genet. 198:255-262; Kondorosi et al., 1984; Innes et al. (1985) Mol Gen. Genet. 201:426-432; Kondorosi et al. (1985) Nitrogen Fixation Research Progress, Evans et al. (eds.) Martinus Nijhoff, Dordrecht, Netherlands, pp. 73-78; Long et al. (1985) ibid., pp. 87-93; Downie et al. (1985) ibid., pp. 95-100; Rolfe et al. (1985) ibid., pp. 79-85; Schofield and Watson (1985) ibid., p. 125).
Three "common" Sym plasmid encoded nodulation genes have been identified in R. meliloti (Torok et al. (1984) Nucleic Acids Res. 12:9509-9524; Jacobs et al. (1985) J. Bacteriol. 162:469-476), R. leguminosarum (Rossen et al. 1984) Nucleic Acids Res. 12:9497-9508) and R. trifolii (Schofield and Watson (1986) Nucleic Acids Res. 14:2891-2903; Rolfe et al. (1985) Nitrogen Fixation Research Progress, Evans et al. (eds.), Martinus Nijhoff, p. 79-85; Schofield and Watson, ibid., p. 125; Schofield Ph.D. Thesis (1984) Australian National University, Canberra, Australia). These genes, designated nodA, B and C, are associated with the early stages of infection and nodulation and are functionally and structurally conserved among fast-growing rhizobia. In R. meliloti, R. leguminosarum and R. trifolii, the nodA B and C genes are organized in a similar manner and are believed to be coordinately transcribed as a single genetic operon. Bacteria having mutations in these genes fail to induce visible nodules on host legumes (nod-) and in some cases even fail to induce root hair curling (hac-) which is prerequisite for infection. The DNA region adjacent to nodA (5'- from the start of nodA) in R. meliloti was reported to be involved in early nodulation function (Torok et al., 1984). The region adjacent to the nodABC operon in fast-growing rhizobia has been shown to contain an open reading frame, designated nodD. Homologous, similarly located nodD genes have been identified and sequenced in R. meliloti (Eglehoff et al. (1985) DNA 4:241-248; Gottfert et al. (1986) J. Mol. Biol. 191:411-420), R. leguminosarum (Shearman et al. (1986) EMBO J. 5:647-652; Downie et al. (1985) Mol. Gen. Genet. 198:255-262) and R. trifolii (Schofield and Watson, 1985 and 1986; Rolfe et al., 1985; Schofield, 1984). Mutations in nodD of R. meliloti are reported to be complemented by the nodD gene of R. trifolii (Fisher et al. (1985) Applied Environ. Microbiology 49:1432-1435). Comparison of the DNA sequences of nodD genes and the deduced amino acid sequences of the nodD proteins confirm the existence of significant sequence conservation of these genes among fast-growing rhizobia.
NodD mutants of the various Rhizobium strains do not, however, display the same nodulation phenotypes. NodD mutants of R. leguminosarum (Downie et al. (1985) Mol. Gen. Genet. 198:255-262) and R. trifolii (Schofield et al.
(1983) Mol. Gen. Genet. 192:459-465) are reported to display unequivocal Nod- phenotypes. In contrast, R. meliloti nodD mutants are reported to have either a "leaky" Nod- phenotype characterized as nod- only in some trials or a delayed nodulation phenotype (Jacobs et al. (1985) J. Bacteriol. 162:469-476; and Gottfert et al. (1986) J. Mol. Biol. 191:411-420). In fact, two distinct nodD genes have been found in R. fredii USDA 191, mutants of which display distinct phenotypes (Appelbaum et al., European Publication No. 0211662 R. fredii nodD-rl, hereinafter designated nodD-2, mutants are delayed in soybean (Glycine max cv. Peking) nodulation and affected in exopolysaccharide synthesis, while nodD-r2, hereinafter designated nodD-1, mutants are nod- on siratro. R. fredii nodD-r2 has been renamed nodD-1 because this gene complements the nodD gene of R. trifolii that is adjacent to the nodABC operon. R. fredii nodDr-1 has been renamed nodD-2 for consistency.
It appears that R. meliloti strains also carry multiple nodD-like genes (Honna et al. (1985) in Nitrogen Fixation Research Progress, Evans et al. (eds.), Martinus Nijhoff Pub., Dordrecht, Netherlands, p. 120; Honna & Ausubel (1986) in Molecular Genetics of the Plant-Microbe Interaction, Verma et al. (eds.), Martinus Nijhoff Pub., Dordrecht, Netherlands, p. 223; Kondorosi et al. (I986) ibid., p. 217; Gottfert et al. (1986)). At least three nodD hybridizing regions have been located on the R. meliloti Sym plasmid; designated nodD-1 (adjacent to nodABC), nodD-2 and nodD-3. NodD-3 mutants appear to retain wild-type nodulation phenotype, while nodD-2 mutants display nodulation delays similar to nodD-1 mutants. Interestingly, double mutants in both nodD-1 and nodD-2 are reported to remain nod+, but display a more severe delay in nodulation. Interspecific hybridization studies using a nodD specific probe reported by Kondorosi et al. (1986) and Rodriguez-Quinones et al. (1987) Plant Mol. Biol. 8:61-75, also indicate the presence of two nodD-like genes in R. trifolii. R. phaseolii and a broad host range Rhizobium sp. MPIK3030.
In contrast to the fast-growing rhizobia, Sym plasmids have not been associated with nodulation by the Bradyrhizobium strains. The nitrogenase and nodulation genes of these organisms are believed to be encoded on the chromosome. Marvel et al. (1984) in Advances in Nitrogen Fixation Research, Veeger and Newton (ed.) Nijhoff/Junk, The Hague, Netherlands; and (1985) Proc. Natl. Acad. Sci. 82:5841-5845, have shown that a strain of Bradyrhizobium sp. (Parasponia) contains genes, associated with early nodulation, which can functionally complement mutations in R. meliloti gene mutants and which hybridize to the nodABC genes of R. meliloti. The presence of nodD gene(s) in this strain was not reported.
Noti et al. (1985) Proc. Natl. Acad. Sci. USA 82:7379-7383 reported the isolation and characterization of nodulation genes from a strain of Bradyrhizobium sp. (Viona). DNA regions within a cloned segment of this strain of Bradyrhizobium were associated with nodulation functions using Tn5 mutagenesis and the cloned DNA was reported to complement nodC mutants of R. meliloti.
Russell et al. (1985) 164:1301-1308 report the isolation of DNA regions encoding nodulation functions in strains of B. japonicum. The isolated DNA region was reported to show strong homology to nod regions of R. meliloti and R. leguminosarum, and to functionally complement a nod-mutation in R. fredii. No sequence or transcript mapping of the cloned DNA was provided.
The precise biochemical role of the nod genes and their products in nodule development is unknown. Attempts to isolate nod gene mRNA and protein products from free-living Rhizobium have been unsuccessful (Kondorosi et al. 1984). Protein products of nod genes have, however, been obtained by fusion of nod genes to strong E. coli promoters (Schmidt et al. (1984) EMBO J. 3:1705-1711; John, M. et al. (1985) EMBO J. 4:2425-2430) or in an E. coli in vitro transcription/translation system (Downie et al. (1985) Mol. Gen. Genet. 198:255-262). Schmidt et al. 1984 report the expression in E. coli minicells of several polypeptides encoded in the R. meliloti common nod region. Three polypeptides of 23, 28.5 and 44 kd, respectively, were mapped to the nod gene cluster. The 44 kd protein maps to a region of DNA strongly conserved among fast-growing rhizobia. John et al., 1985 identify the 44 kd protein as the product of the nodC gene. A fourth polypeptide product of 17.5 kd is mapped to the region of the nod gene in R. meliloti. Downie et al. 1985 report the production of the presumptive nod gene products of R. leguminosarum by an in vitro translation/transcription system. Four polypeptides having molecular weights of 48, 45, 36 and 34 kd were reported to be the products of the nod genes. The 34 kd and 36 kd polypeptides are described as originating from a single gene and are reported to be the products of nodD.
The establishment of nitrogen-fixing nodules is a multistage process involving coordinated morphological changes in both bacterium and plant, so it is expected that the rhizobial nodulation genes are under precise regulatory control. It has been suggested that an exchange of signals between plant and bacterium is requisite for mutual recognition and coordination of the steps of infection and nodulation development (Nutman, P.S. (1965) in Ecology of Soil Borne Pathogens, eds. F.K. Baker and W.C. Snyder, University of California Press, Berkeley, pp. 231-247; Bauer, W.D. (1981) Ann. Rev. Plant Phys. 32:407-449; and Schmidt, E.E. (1979) Ann. Rev. Microbiol. 33:355-376). For example, root exudates have been linked to control of nodulation. Exudates have been reported to both stimulate (Thornton (1929) Proc. Royal Soc. B 164:481; Valera and Alexander (I965) J. Bacteriol. 89:113-139; Peters and Alexander (1966) Soil Science 102:380-387) and inhibit (Turner (1955) Annals Botany 19:149-160; and Nutman (1953) Annals Botany 17:95-126) nodulation by rhizobia.
Reports on the regulation of the nod genes of R. meliloti (Mulligan and Long (1985) Proc. Natl. Acad. Sci. USA 82:6609-6613) and R. leguminosarum (Rossen et al. (1985) EMBO J. 4:3369-3373; Shearman et al. (1986) EMBO J. 647-652) and R. trifolii (Innes et al. (1985) Mol. Gen. Genet. 201:426-432) have appeared. All of these studies report that nodC is expressed at very low levels in free-living R. meliloti, but is induced in the presence of plant exudate. Shearman et al. 1986 reports that in addition, nodF of R. leguminosarum is induced in the presence of plant exudates and Innes et al. 1985 reports that several other R. trifolii nod genes including nodFE and genes of region IV are induced by legume exudate. In all studies, nodD expression is reported to be constitutive and not to be inducible by root exudates.
In R. meliloti and R. leguminosarum, nodD is reported to be necessary in addition to plant factors for expression of the nodABC genes (Downie et al. (1985) Mol. Gen. Genet. 198:255-262; Mulligan and Long, 1985; Rossen et al., 1985). More recently, Shearman et al. (1986) EMBO J. 5:647-652, have reported that nodD is also required, in addition to plant factors for induction of nodF. Similarly, in R. trifolii , the nodABC genes are unable to confer root hair curling, that is prerequisite for nodulation, in the absence of the nodD gene (Schofield, Ph.D. Thesis (1984) Australian National University, Canberra). These results indicate that the nodD gene product has a regulatory function in fast-growing rhizobia in the expression of several other nod genes. The mechanism by which nodD regulates the expression of other nod genes is not fully understood, but may involve the initial interaction of nod directly or indirectly with legume exudate factors followed by binding of an altered nodD to DNA sequences in the promoter regions of the legume exudate inducible nod genes. The role of the multiple copies of nodD genes in a single strain in the regulation of nodulation is not yet fully understood.
A highly conserved nucleotide sequence has been described in the promoter regions of several legume exudate-inducible nod genes. This sequence precedes the R. trifolii nodABC and nodFE genes and the R. meliloti nodABC genes (Schofield and Watson (1986) Nucleic Acids Research 14:2891-2904). The sequence has also been identified in the promoter region of the nodABC genes in the slow-growing Bradyrhizobium sp. (Parasponia) Scott (1986) Nucleic Acids Res. 14:2905-2919). This sequence is believed to function in the regulation of expression of nod genes by chemical factors in legume exudate (Schofield et al., Innes et al., (1985 Mol. Gen. Genet. 201:426-432), possibly as a DNA binding site for nodulation regulatory proteins.
Specific components of legume exudates that act to induce nodulation gene expression in several species of Rhizobium have recently been identified. In addition, a number of compounds related in structure to the inducer components found in exudates have also been identified as inducers of Rhizobium nodgenes.
Peters et al. (1986) Science 233:977-980 identified luteolin (3',4',5,7-tetrahydroxyflavone) as the component of alfalfa exudates that induces nodABC expression in R. meliloti. Nod gene induction was assayed as .beta.-galactosidase expressed from a lacZ gene which had been fused to the nodC gene of R. meliloti. In this gene fusion, the lacZ structural gene was placed under the regulatory control of the nodABC promoter and its associated nod-box regulatory sequence. A number of chemical compounds structurally related to luteolin were also assayed for nod-gene induction in this system including several flavones, flavanones and flavanols. Of those compounds tested, only apigenin was found to induce the R. meliloti nodgene. Apigenin was found to be a much weaker inducer than luteolin.
Using similar nod-lacZ fusions in several nod genes, Redmond et al. (1986) Nature 323:632-635 reported the identification of three clover exudate components that induced expression of R. trifolii nodgenes: 4',7- dihydroxyflavone (DHF), geraldone (3'-methoxy DHF) and 4'-hydroxy-7-methoxyflavone. In related work, Rolfe et al. European Publication No. 0245931, a number of substituted flavones, flavanols and flavanones were identified as R. trifolii nod gene inducers including luteolin and naringenin. Induction activity was reported to be confined to molecules having the flavone ring structure, in particular coumestrol and the isoflavones daidzein and formononetin were not active for R. trifolii nod gene induction.
Two of the nodulation gene inducers of R. leguminosarum from pea exudate were identified as eriodictyol (3',4',5,7-tetrahydroxyflavanone) and apigenineriodict 7-0-glucoside by Firmin et al. (1986) Nature 324:90-92. Apigenin, hersperitin and naringenin, in addition to other flavones and flavanones, were also found to be active as inducers. The isoflavones daidzein, genistein and kaempferol were reported to be antagonists which strongly inhibited the activation of nod genes by inducers.
Zaat et al. (1987) J. Bacteriol. 169:198-204 characterized a R. leguminosarum nodulation gene inducer from Vicia sativa exudate as "flavonoid in nature, most likely a flavanone." Although the exudate component was not identified, naringenin, eriodictyol, apigenin and luteolin were reported to be strong nodgene inducers; 7-hydroxyflavone, a somewhat weaker inducer, and chrysin and kaempferol were weak or poor inducers. Among others, the isoflavones daidzein, genistein and prunetin were reported to be inactive.
Kosslak et al. U.S. Pat. Application Ser. No. 035,516, filed Apr. 7, 1987, now abandoned, reports the identification of chemical factors which induce B. japonicum nod gene expression. The isoflavones daidzein and genistein were identified as components of soybean exudates which induced nodgene expression. Additionally, several other isoflavones including 7-hydroxyisoflavone, 5,7-dihydroxyisoflavone, biochanin A; formononetin and prunetin, as well as the flavone apigenin, the flavonol kaempferol and coumestrol were found to induce nodgene expression. These results were unexpected in that isoflavones were found not to induce nod gene expression in Rhizobium strains and in fact isoflavones and several other of the Bradyrhizobium japonicum nod gene inducer compounds had been reported to be antagonists of nod gene induction at least in Rhizobium leguminosarum (Firmin et al., 1986). The identification of the chemical factors which induce the nodulation genes of Bradyrhizobium sp. Parasponia has not been reported.
Recently Horvath et al. (1987) EMBO J. 6:841-848 reported a comparison of nodgene induction in R. meliloti and Rhizobium MPIK3030, a derivative of Rhizobium NGR234 which is a broad host range nodulating strain which nodulates siratro, among others. It is reported that the MPIK3030 nodD-1 gene induces expression of nodulation genes by interacting with plant factors from the host siratro as well as the non-host alfalfa. In contrast, R. meliloti nodD genes interact only with alfalfa exudate. R. meliloti transconjugates carrying the nodD-1 from MPIK3030 effectively nodulate siratro; however, MPIK3030 transconjugants carrying the R. meliloti nodD-1 do not nodulate alfalfa. A chimeric nodD gene having the 5' end of nodD-1 of MPIK3030 and the 3' end of R. meliloti nodD-1 was inserted into a R. meliloti nodD-1/nodD-2 mutant. The R. meliloti mutant transconjugant carrying the chimeric gene nodulated alfalfa normally. In contrast, an MPIK3030 mutant transconjugant carrying the chimeric gene did not nodulate siratro. These results indicate that nodD regulation of nodulation genes mediated by interaction with plant factors is host specific presumably by the selective interaction of nodD proteins with certain plant factors. The results also indicate that the carboxy end of the nodprotein, which is the more divergent region among the nodD proteins, is functional in the specific plant factor interaction which can result in host specificity of nodulation. Horvath et al. note that the apparent specificity of the interaction between plant factors and nodD protein may result from an enhanced affinity of certain plant factors for a nodD protein or alternatively from a decreased susceptibility of a nodD to inhibiting compounds found in exudates (Firmin et al., 1986).